Fibroblast activation protein (fap) targeted imaging and therapy in fibrosis

ABSTRACT

Excessive deposition of extracellular matrix is a hallmark of Idiopathic pulmonary fibrosis (IPF), it is advantageous to target the cells and the mechanisms associated with this process. By targeting myofibroblasts (specialized contractile fibroblasts) that are key for the development of IPF with drugs conjugated with fibroblast activation protein (FAP), this technology helps minimize the production of extracellular matrix in the lungs and provides a new treatment option for patients diagnosed with IPF.

FIELD OF INVENTION

This disclosure provides a conjugate and method of using thereof toimage and/or treat idiopathic pulmonary fibrosis (IPF). Specifically,Fibroblast active protein (FAP) targeted imaging agent or therapeuticdrug is delivered to IPF to either guide the diagnosis of IPF orsignificantly reduces IPF pathological extracellular matrix deposition.

BACKGROUND

Fibrotic diseases constitute a major health problem worldwide affectinga large number of individuals.

Under pathologic circumstances, the normal tissue repair reactionescapes the homeostatic regulatory mechanisms and evolves into anuncontrolled fibrotic process characterized by the progressiveover-production of extracellular matrix which disrupts the normal organarchitecture and ultimately leads to organ failure.

Virtually every organ in the human body can be affected by physiologicand pathologic fibrotic reactions, but the most commonly affected organsare the lungs, kidneys, liver, skin, heart, and bladder. For example,fibrosis of the liver represents a paradigm for this disease, as it maybe reversible at early stages but become irreversible as it progressesto cirrhosis, resulting in liver cancer in addition to end stagedisease. It has multiple potentially preventable etiologies; theyinclude HBV and HCV infection, obesity, alcoholism, and aflatoxin amongothers; each presents opportunities for and serious barriers to primaryand/or secondary prevention. For many other fibrotic diseases, theunderlying etiologies are less clear, although many are associated withchronic production of proteolytic enzymes, fibrogenic cytokines, growthfactors, and angiogenic factors, presumably secondary to triggeringirritants (e.g., radiation, chronic infections, toxins). Others arecongenital or associated with autoimmunity. For fibrosis of all types,the point of irreversibility and the molecular mechanisms by which itoccurs are not well defined. Organ failure is the end-result ofuncontrolled fibrosis. Treatment of fibrotic diseases in these organs isnecessary to prevent the eventual organ failure and morbidity.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic disease oflungs. It is believed to be caused by repetitive environment injury tothe lining of the lungs and resulting abnormal wound-healing responses.When tissues of lung experience prolonged activation of wound healingresponses, the result usually is permanent scarring, organ malfunctionand more significantly, death.

It is estimated that there are about 128,000 people living with IPF,with 48,000 new cases diagnosed annually in US alone. Among them, about40,000 IPF patients die each year. Median survival age after diagnosisis approximately 2-3 years. Owing to the widespread and multi-organoccurrence of fibrotic diseases, it is difficult to determine theirtotal incidence, although it has been estimated that as high as 45% ofthe mortality in Western developed countries is caused by theircollective occurrence.

Until recently, there have been no curative therapies for IPF, withtreatments options limited to lung rehabilitation and oxygen therapy.

Currently, FDA approved two drugs in 2014 for the treatment of IPF:pirfenidone and nintedanib. However, both drugs show limited andinconsistent efficacy in IPF patients. Very recently, several kinaseinhibitors have been introduced into human clinical trials with the hopethat they might block essential steps in the fibrotic process, howevertheir on targeted activities against the same enzymes in healthy tissueshave raised concerns regarding systemic toxicities.

In more advanced cases, lung transplantation can be a final option, butfinding an HLA match is often difficult and avoiding transplantrejection can be challenging.

Therefore, an urgent need for therapies that can slow IPF progressionexists.

SUMMARY OF THE INVENTION

This disclosure provides a conjugate to target Fibroblast ActivationProtein (FAP) expressing cells in fibrotic lung diseases. The conjugatecomprises a targeting ligand to FAP (TL), a linker (L) and an effector(E), wherein the TL has a molecular weight below 10,000, the L is anon-releasable linker when the effector is an imaging agent or aradioactive therapeutic agent; the L is a releasable linker when theeffector is a therapeutic drug. The linker is selected from the groupconsisting of pegylated, alkyl, sugar or peptide based dual linker.

In some preferred embodiment the aforementioned imaging agent is afluorescent molecule.

In some preferred embodiment the aforementioned conjugate comprises thestructure of FAPL-FITC below:

In some preferred embodiment the aforementioned fluorescent dye is anear infrared dye and the conjugate has the structure of

In some preferred embodiment the aforementioned florescent moleculecomprising the structure of

In some preferred embodiment the aforementioned effector is a PETimaging agent.

In some preferred embodiment the aforementioned PET imaging agentcomprises the structure of

In some preferred embodiment the aforementioned effector is a 99mTcimaging agent comprising a DOTA, NOTA, TETA or NODAGA chelating agent

In some preferred embodiment the aforementioned 99mTc imaging agentcomprises the structure of.

In some preferred embodiment the aforementioned radioactive therapeuticagent contains the structure of

In some preferred embodiment the aforementioned TL is a small moleculecomprising the structure of

-   -   wherein x is

-   -   wherein R₁ and R₂ are the same or different, and are each        independently selected from the group consisting of hydrogen,        halogen and C₁-C₄ alkyl;    -   R₃ is a C₁-C₄ alkyl, nitrile, or isonitrile;    -   R₄ is H or —CH₃    -   R₅ and R₆ are the same or different, and are each independently        selected from the group consisting of hydrogen, halogen, and        C₁-C₄ alkyl,    -   R₇-R₉ are the same or different, and are each independently        selected from the group consisting of hydrogen, methoxy,        halogen, CF₃ and C₁-C₄ alkyl.

In some preferred embodiment the aforementioned R₁ and R₂ is a halogen.

In some preferred embodiment the aforementioned each of R₁ and R₂ isfluorine.

In some preferred embodiment the aforementioned effector is a kinaseinhibitor for VEGFR1, VEGFR2, VEDFR3, FGFR1, FGFR2, or PDGFR.

In some preferred embodiment the aforementioned effector is a kinaseinhibitor for FAK or ROCK.

In some preferred embodiment the aforementioned effector is an SMADinhibitor.

In some preferred embodiment the aforementioned effector is a cytotoxicagent.

In some preferred embodiment the aforementioned effector is a PI-3kinase inhibitor.

This disclosure further provides a PI-3 Kinase inhibitor comprising thestructure of (PI3KI1).

In some preferred embodiment the aforementioned conjugate having thestructure of

In some preferred embodiment the aforementioned PI-3 kinase inhibitorcomprising the structure below:

wherein X can be any of the following

In some preferred embodiment the aforementioned targeting ligand to FAPhas a binding affinity to FAP in the range between about 1 nM and about10 nM.

This disclosure further provides a method of diagnosing IPF in asubject, comprising the following steps:

-   -   obtaining the lung tissue from the subject, wherein said tissue        may or may not express FAP in fibroblast cells;    -   providing to the tissue with a conjugate of TL-L-I, wherein TL        is a targeting ligand A conjugate to target Fibroblast        Activation Protein (FAP) expressing cells in fibrotic lung        diseases, wherein said TL has a molecular weight below 10,000,        said L is a non-releasable linker, said I is an imaging agent;        and    -   identifying imaging illustrated fibroblast cells as FAP        expressing activated fibroblast cells as the hallmark of IPF.

In some preferred embodiment the aforementioned TL is a small moleculehaving the structure of

wherein x is

wherein R₁ and R₂ are the same or different, and are each independentlyselected from the group consisting of hydrogen, halogen and C₁-C₄ alkyl;

-   -   R₃ is a C₁-C₄ alkyl, nitrile, or isonitrile;    -   R₄ is H or —CH₃;    -   R₅ and R₆ are the same or different, and are each independently        selected from the group consisting of hydrogen, halogen, and        C₁-C₄ alkyl;    -   R₇-R₉ are the same or different, and are each independently        selected from the group consisting of hydrogen, methoxy,        halogen, CF₃ and C₁-C₄ alkyl.

This disclosure further provides a method of treating IPF in a subject.The method comprising the steps of:

-   -   providing to the IPF patient cells with a pharmaceutically        effective amount of conjugate of TL-L-D, wherein TL is a        targeting ligand to FAP that has a molecular weight below        10,000, L is a releasable linker and D is a therapeutic drug        that has pan PI-3Kinase inhibitory effect; and    -   monitoring lung tissue extracellular matrix deposit amount        reduction upon the treatment of TL-L-D.

In some preferred embodiment the aforementioned TL is a small moleculehaving the structure of structure of

wherein x can be

-   -   wherein R₁ and R₂ are the same or different, and are each        independently selected from the group consisting of hydrogen,        halogen and C₁-C₄ alkyl;    -   R₃ is a C₁-C₄ alkyl, nitrile, or isonitrile;    -   R₄ is H or —CH₃;    -   R₅ and R₆ are the same or different, and are each independently        selected from the group consisting of hydrogen, halogen, and        C₁-C₄ alkyl,    -   R₇-R₉ are the same or different, and are each independently        selected from the group consisting of hydrogen, methoxy,        halogen, CF₃ and C₁-C₄ alkyl.

In some preferred embodiment the aforementioned D has the structure offollowing:

wherein X can be any of the following:

In some preferred embodiment the aforementioned method uses a conjugatethat reduces collagen I deposits on activated fibroblast cells.

In some preferred embodiment the aforementioned method uses a subjectthat is a mouse IPF model induced by intratracheal administration ofbleomycin at about 0.75 u/kg for consecutive 10 days.

In some preferred embodiment the aforementioned method administering aconjugate at about 0.2-10 umol/kg to the mouse IPF model for consecutive10 days and the conjugate is FAPL_PI3KI1 with the structure of.

In some preferred embodiment the aforementioned method monitorsextracellular matrix collagen I.

In some preferred embodiment the aforementioned method reduces thehydroxyproline production of fibroblast cells.

In some preferred embodiment the aforementioned method uses a subject ofa mouse IPF model induced by silica or radiation.

These and other features, aspects and advantages of the presentinvention will become better understood with reference to the followingfigures, associated descriptions and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Design and Synthesis of FAPL-FITC conjugate for analysis of FAPtargeting.

FIG. 2. Binding and Internalization of FAPL-FITC by Human FibroblastCell Line

FAPL-FITC targets human FAP in HLF1 cells. A) Live cell imaging ofFAPL-FITC internalization. confocal microscopy images of HLF1-hFAP cellsincubated with FAPL-FITC (10 nM), FAP staining is shown in green.Spatial localization of FAPL-FITC in HLF1-hFAP cell line at 0 min (a),and at 30 min (d). Colocalization of FAP and endosomes: HLF1-hFAP cellsincubated with FAPL-FITC followed by staining for endosomes (Rab7a-RFP;red) and nucleus (DRAQ5; blue). The merge of the 3 images is shown onthe right (c, and f). Yellow indicated colocalization of FAP withendosomal markers.

FIG. 3. Binding of FAPL-FITC by Human IPF Patient Cell Line

αSMA and FAP predominantly expressed in IPF lung fibroblasts andtargeted by FAP-FITC. (A) IPF fibroblasts and non IPF control fibroblastwere seeded and stained with antibody against FAP or aSMA. (B) IPF cellswere incubated with FAPL-FITC (10 nM) and analyzed by flow cytometry.

FIG. 4. Design and Synthesis of PI3KI1 and FAPL-PI3KI1

Design and the synthesis of a novel pan-Pi3K inhibitor. (A) Putativebinding of PI3KI1, based on the crystal structure of Pi3Kg (PDB code:3L08). (B) docking scores of compounds docked. (C) Synthetic scheme ofPI3KI1

FIG. 5. Synthesis of novel FAP targeted pan-Pi3K inhibitor(FAPL_PI3KI1).

FIG. 6. Evaluation of Myofibroblast Inactivation with FAP-Targeted PI-3Kinase Inhibitor In Vitro.

PI3KI1 inhibits Akt phosphorylation, proliferation, collagen secretionand collagen gel contraction in HLF-1 fibroblasts. (A) PI3KI1 structure.(B) Confluent HLF-1 fibroblasts were stimulated with TGFβ (1 ng/mL) withindicated PI3KI1 or OMIPALISIB and lysates collected for Westernblotting. PI3KI1 inhibits TGF-β (1 ng/ml for 24 hr) induced AKTphosphorylation with IC₅₀ 1.4 nM. Collagen 1 and αSMA expression wassuppressed by 100 nM PI3KI1, but pSMAD2 was not affected by PI3Kinhibitors. (C) MTT assay and Caspase 3 and 7 activity (Supplementarydata) show PI3KI1 inhibited HLF-1 proliferation at >100 nM. (D) HLF-1fibroblasts were stimulated with TGFβ (1 ng/mL) for 3 days and level ofsecreted collagen in culture medium was determined by Sirius Redstaining. (E) PI3KI1 (100 nM for 12 hours) disrupted fibroblastsreorganization and contraction, characteristics of activated fibrosis,by collagen gel contraction assay. Data are analysis by t-test; *p<0.05,**p<0.01.

FIG. 7. Ex Vivo IPF Patient Cell Line Data

PI3KI1 and PI3KI1-FAPL suppressed TGFβ-induced collagen production inIPF fibroblasts. (B) Confluent IPF fibroblasts were stimulated for 48 hwith TGFβ (1 ng/mL) with increasing concentrations of OMIPALISIB orPI3KI1 or PI3KI1-FAPL and Collagen I expression was assayed by molecularcrowding assay with high content image analysis. Data are expressed asrelative fluorescent intensity over TGFβ treated IPF fibroblasts (C) andcell counts obtained from DAPI counterstaining (D).

FIG. 8. Evaluation of FAP Targeting in Mouse Fibroblast Cell Line InVitro

FAPL-FITC targets mouse FAP in mouse fibroblast cells. Fluorescencebinding affinity study of FAPL_FITC in HLF-hFAP cells. (C) Binding ofFAPL_FITC to TGFb induced mouse NIH-3T3 cell line.

FIG. 9. Evaluation of FAP Targeting of IPF Lung in Mouse Model of IPF InVivo

(I) Optical imaging of experimental lung fibrosis in mice with FAP-atargeted NIR dye, open body (A), biodistribution (B). Representativeoptical images in PBS administered (a, d) and bleomycin (0.75 u/Kg)administered mice at day 14 in the presence (b, e) or in the absence (c,f) of 100-fold excess of the FAP ligand. Images were acquired after 4hours post injection of 5 nmoles of the FAP_S0456. The color barindicates radiant efficiency (low, 2.7×108; high, 5.9×108).Biodistribution: 1) heart, 2) lung, 3) spleen, 4) muscle, 5) stomach, 6)small intestine, 7) large intestine, 8) liver, and 9) kidneys. (II)Representative optical images of the lungs, with FAP_S0456 of PBSadministered healthy control (a), 7 days (b), 14 days (c), and 21 days(d) after bleomycin administration, and Quantitative fluorescenceintensity change in the lungs over time. For optical imaging all theimages were acquired and compared under the same condition, after 4hours post injection of 5 nmoles of the FAP_S0456. The color barindicates radiant efficiency (low, 1.7×10⁸; high, 3.6×10⁸). (III)Representative CT images of the lungs, PBS administered healthy control(a), 7 days (b), 14 days (c), and 21 days (d) after bleomycinadministration.

FIG. 10. Evaluation of Myofibroblast Inactivation with FAPL-TargetedPI-3 Kinase Inhibitor In Vivo

Therapy with FAP-a targeted Pi3K inhibitor in bleomycin treated mice.(A) Schematic representation of the experimental protocol for induction,treatment and examination of pulmonary fibrosis in a mouse model. (B)Survival probability of the FAPL_PI3KI1 treated mice over the salinecontrol. (C) Hydroxyproline content (ug/lung) of control (saline),bleomycin treated with and without FAPL_PI3KI1 treatment at day 21. (D)Body weight change

FIG. 11. Characterization of pulmonary fibrosis in mice over time,post-bleomycin (0.75 u/Kg) administration. (B) Hydroxyproline analysisof disease progression in bleomycin treated mice with saline treatedmice at day 7, day 14, and day 21

FIG. 12. TGF-β (1 ng/ml for 24 hr) induced FAP and aSMA expression inhuman lung fibroblast cell line (HLF-1). HLF-1 cells were 0.4% serumstarved for 12 hr and then stimulated by TGF-β (1 ng/mL) for 24 hr. FAPLand αSMA expression were detected by immunofluorescence analysis.

FIG. 13. Chemical structure of Compound 3.

FIG. 14. Chemical structure of Compound 8.

FIG. 15. Chemical structure of Compound 5.

FIG. 16. Chemical structure of FAPL_PI3KI1.

FIG. 17. General method of making synthetically derived compounds.

FIG. 18. Caspase 3 and 7 activity show PI3KI1 inhibited HLF-1proliferation at >100 nM.

FIG. 19. Radio-imaging of experimental lung fibrosis in mice with FAPtargeted ^(99m)TC conjugate. 19A shows FAP targeted TC in variousorgans, and 19B shows FAP targeted ^(99m)TC in the lungs.

DETAILED DESCRIPTION

While the concepts of the present disclosure are illustrated anddescribed in detail in the figures and the description herein, resultsin the figures and their description are to be considered as exemplaryand not restrictive in character; it being understood that only theillustrative embodiments are shown and described and that all changesand modifications that come within the spirit of the disclosure aredesired to be protected.

Unless defined otherwise, the scientific and technology nomenclatureshave the same meaning as commonly understood by a person in the ordinaryskill in the art pertaining to this disclosure.

IPF is a lethal, chronic, progressive disease, and increases with age,particularly in individuals over the age of 50. In US, IPF kills as manypeople (about 40,000 per year) as breast cancer, with most patientsdying within 3-5 years of diagnosis.

Until recently, there have been no curative therapies for IPF, withtreatments options limited to lung rehabilitation and oxygen therapy.However, in 2014, two new drugs, pirfenidone and nintedanib, wereapproved by the FDA for treatment of IPF. Unfortunately, both showlimited and inconsistent efficacy, primarily retarding diseaseprogression but not leading to stable resolution of the disease.

Very recently, several kinase inhibitors have been introduced into humanclinical trials with the hope that they might block essential steps inthe fibrotic process, however their on-target activities against thesame enzymes in healthy tissues have raised concerns regarding systemictoxicities.

In more advanced cases, lung transplantation can be a final option, butfinding an HLA match is often difficult and avoiding transplantrejection can be challenging.

Given the difficulty of finding an enzyme or pathway that is uniquelyrequired for pathologic fibrosis, we have undertaken to identify amolecular marker that is primarily expressed on fibrosis-producing cellsthat might be exploited for the targeted delivery of drugs to thesecells.

In response to stimulation by activated immune cells such as macrophagesand T cells, fibroblasts become activated to myofibroblasts that thenaccumulate in areas called fibroblast foci where they produce thecollagen that causes the fibrosis.

These myofibroblasts are readily distinguished from nonpathologicfibroblasts by their expression of a transmembrane protein, fibroblastactivation protein (FAP) that is critical for the process of collagenremodeling.

The unique expression of FAP on myofibroblasts provides a marker thatcan conceivably be exploited for the targeted delivery of drugs tomyofibroblasts.

In this study, a low molecular weight ligand specific for fibroblastactivation protein (FAP) was synthesized and evaluated for binding to anFAP-expressing human lung fibroblast cell line transfected with FAP(HLF1-FAP). The potency of a novel PI-3 kinase inhibitor targeted withthe FAP ligand (FAPL_PI3KI1) in suppressing collagen synthesis andsecretion by both HLF1-hFAP cell line and IPF patient cell lines wasalso examined. Specific targeting of the fibrotic lungs was thenevaluated in vivo using an FAPL-targeted NIR dye (FAP-S0456). Finally,the ability of the targeted inhibitor, FAPL-PI3KI1, to reduce collagendeposition and fibrosis in the lungs of bleomycin-induced mice wasassessed.

We have shown that FAP ligand binds to FAP in HLF1-FAP cells with ˜3 nMaffinity. FAPL-PI3KI1 was observed to potently inhibit collagensecretion/synthesis (IC₅₀=<10 nM) in both HLF1-hFAP and IPF patient celllines. NIR imaging of bleomycin-induced mice showed specific targetingof FAPL-S0456 NIR dye to fibrotic lungs with good competition. In vivotherapy with FAPL-PI3KI1 demonstrated increased survival and decreasedhydroxyproline/collagen production compared to PBS-treated control miceinduced to develop experimental lung fibrosis. These findingsdemonstrate, 1) FAPL-S0456 can target the fibrotic lungs with goodspecificity, 2) targeted delivery of Pi3K inhibitor (FAPL-PI3KI1) may bebeneficial in treating lung fibrosis, as well as other diseases that arecharacterized by pathological inflammation and fibrosis.

Various embodiments are provided to exemplify the making and using ofFAP targeting conjugates and their promising effects on reducing oreliminating IPF symptoms, such as reduced collagen I production. Basedon the expression pattern of FAP on various cancer tissues, it iscontemplated that FAPL conjugated warhead can be used on other diseasemodels as well, as long as the therapeutic drug conjugated to FAPL isspecific and effective for the disease.

It is established that FAP protein expression is restricted, occurringat high levels on mesenchymal cells during embryogenesis then repressedshortly after birth, and its expression is upregulated on activatedfibroblast in conditions associated with wound healing, cancer andfibrosis. From the examples below, a person skill in the art willappreciate that the expression pattern of FAP qualifies it as a goodmarker for the targeted delivery of drugs or other effectors to fibroticcells, a hall mark of fibrosis.

Based on the data presented in the following examples, a FAP ligandtargeted PI-3 kinase therapy can be primarily applied to adults at timeswhen they are not recovering from a serious lung or other tissue trauma,one would not expect to encounter significant off target toxicity withthe use of this drug.

It is also recognized that the fibrotic process share some similaritiesamong different types of fibrosis, therefore, a targeted drug that mightinactivate a myofibroblast and reprogram it to become a quiescencefibroblast might also prove useful in treating fibrosis of liver,kidney, heart, skin, and bladder organs. Without being restricted to anytheory, this is because the basic pattern of disease progression inthese several types of fibrosis may involve immune cell activationarising from unknown causes, leading to activation of fibroblasts toform myofibroblasts, thereby triggering the excessive production ofcollagen. Thus, a common feature in essentially all known fibroticdiseases is the production of collagen by the myofibroblasts, whichinvariably expresses FAP regardless of the organ in which the fibrosisoccurs.

While a number of therapeutic warheads could have been selected fordelivery with our FAP targeting ligand, for example, kinase inhibitorsto VEGFR1, VEGFR2, VEDFR3, FGFR1, FGFR2, or PDGFR are potentialcandidates for delivery. Other warheads such as kinase inhibitors forFAK or ROCK, other effectors such as SMAD inhibitor, or cytotoxic agent,are all within the contemplation of this disclosure. In the instantapplication a potent pan PI-3 kinase inhibitor was selected in view ofthe many failures of prior PI-3 kinase inhibitors in the clinic,primarily due to its following advantages: PI-3 kinase pathwayactivation is reported in fibrotic foci, the cardinal lesions in IPF.PI-3 Kinase isoforms exhibit increased expression in IPF tissue andfibroblast lines, with signaling activated downstream of several keyprofibrotic growth factors implicated in IPF, including platelet-derivedgrowth factor and transforming growth factor (TGF)-β1. OMIPALISIBinhibited PI-3 Kinase signaling and functional responses in IPF-derivedlung fibroblasts, inhibiting Akt phosphorylation in IPF lung tissue andBAL derived cells.

Inhibition of PI-3Kinase pathway also effects normal cellular functions,including proliferation, apoptosis and metabolism. A targeted approachis essential to prevent the cumulative toxicity of a pan-PI3K/mTORinhibitor. Although an isoform-specific inhibitor could have beenselected for delivery to the myofibroblasts, a pan-PI3K/mTOR kinaseinhibitor was chosen to overcome any functional redundancy betweenisoforms and blocking potential crosstalk and feedback of compensatorymechanisms through inhibition of three key nodes (PI3K, mTORC1 andmTORC2).²⁵ While such a general PI-3 kinase inhibitor will generally bemore toxic, suppressing all PI-3 kinase dependent processes is not soundesirable, since the myofibroblasts are not essential to normal lungfunction and they generally undergo apoptosis during resolution of thedisease. It is interesting to note the specific accumulation of ourFAPL-targeted fluorescent dye in the fibrotic lungs of thebleomycin-induced mice. Indeed, we have noted that the intensity of dyeuptake correlates with the intensity of the fibrosis. In our studies ofother targeting ligands developed for the treatment of other diseases,we have observed that the same targeting ligand can be used for deliveryof a variety of useful payloads. If the same FAP-targeting ligand can beadapted for delivery of a radio-imaging agent to fibrotic tissues, thepossibility exists that an FAP-targeted imaging agent might be developedfor use in the diagnosis, staging or evaluation of response to therapyin IPF.

Last but not the least, due to PI-3 kinase's broad implications ofdifferent cell functions, particularly in some other disease models, itis contemplated that a given pan PI-3 kinase inhibitor war head can beconjugated to a targeting ligand to a specific disease marker, to exertits inhibitory effect in that particular disease model. For example,invasive bladder cancer treated with PI-3 kinase inhibitor can beimproved with targeted ligand to bladder cancer marker Epidermal growthfactor receptor (EPGR) conjugated with a choice of pan PI-3 kinaseinhibitor. Our platform of pan PI-3 kinase warhead conjugated to adisease specific targeting ligand provides a therapeutic platform modelfor tackling various diseases that have unique surface marker expressionon the diseased tissue.

Experimental Procedure

General. H-Cys(Trt)-2-Cl-Trt resin and protected amino acids werepurchased from Chem-Impex Intl. 2-(Hydroxymethyl)pyridine-5-boronicacid, pinacol ester was purchased from Combi-Blocks.6-bromo-4-iodoquinoline, 2-4-Diflurobenzene-1-sulfonyl-chloride and5-bromo-2-methoxypyridine-3-amine was purchased from ArkPharm. All theother chemicals were purchased from SIGMA-Aldrich and Fisher Scientificand used as received. Thin layer chromatography (TLC) was carried out onMerck silica gel 60 F254 TLC plates. Silica gel column chromatographywas performed using silica gel (60-120 μm particle size). Preparativereverse-phase high performance liquid chromatography (RP-HPLC) wasperformed on a Waters, XBridge™ Prep C18, 5 μm; 19×100 mm column, mobilephase A=20 mM ammonium acetate buffer, pH 5 or 7, B=acetonitrile, systemwith gradients in 30 min, 13 m/min, λ=254/280 nm. The LRMS-ESI (LC-MS)was recorded on Agilent LCMS 1220 system, with Waters, XBridge™ RP18,3.5 μm; 3×50 mm column, mobile phase A=20 mM ammonium bicarbonatebuffer, pH 5 or 7, B=acetonitrile, system with gradients in 12-15 min,0.75 m/min, λ=254/280 nm. The high resolution mass measurements wererecorded on a LTQ Orbitrap XL mass spectrometer utilizing electrosprayionization (ESI).

Synthesis

The FAP ligand (11), FAP-FITC and FAP-S0456 was synthesized following apreviously published procedure. See WO2018111989A1.

2-4-Diflurobenzene-1-sulfonyl-chloride 2 (1 eq) was added slowly to acool solution of 5-bromo-2-methoxypyridine-3-amine 1 (1 eq) in pyridine.Reaction was stirred at ambient temperature for 16 h, at which time thereaction was diluted with water and the solids were filtered off andwashed with copious amounts of water. The precipitate was dried in highvacuum to give compound 3, and was used in the next step without furtherpurification. LRMS-LC/MS (m/z): [M+H]⁺ calcd for C₁₂H₉BrF₂N₂O₃S, 377.9;found 378.9).

A mixture of Bis(Pinacolato)diboron 4 (1 eq), compound 3 (1 eq),Pd(dppf)₂Cl₂ (0.1 eq), KOAc (3 eq) in anhydrous 1,4-dioxane wasdeoxygenated by bubbling nitrogen through it for 10 min. The mixture wasthen heated at reflux for 3 h. After cooling to room temperature, themixture was evaporated under reduced pressure and the residue wasdissolved in EtOAc, washed with water twice and dried over magnesiumsulfate. The crude product was purified by flash chromatography(Hex:EtOAc) to give compound 5. LRMS-LC/MS (m/z): [M+H]⁺ calcd forC₁₈H₂₁BF₂N₂O₅S, 426.1; found 427.1).

6-bromo-4-iodoquinoline 6 (212.37, 0.636 mmol) and2-(Hydroxymethyl)pyridine-5-boronic acid 7 (150 mg, 0.636 mmol) wasdissolved in anhydrous 1,4-dioxane (15 mL). To this was addedPd(dppf)₂Cl₂ (19.9 mg, 0.024 mmol) followed by 2M Na₂CO₃ (2.5 mL). Themixture was then heated at reflux for 6 hrs. After cooling to roomtemperature the solids were filtered off and evaporated. The crudeproduct was purified by flash chromatography (EtOAc:MeOH) to givecompound 8. LRMS-LC/MS (m/z): [M+H]⁺ calcd for C₁₅H₁₁BrN₂O, 314; found315).

PI3 KI1

Compound 5 (136 mg, 0.32 mmol) and compound 8 (110 mg, 0.32 mmol) wasdissolved in anhydrous 1,4-dioxane (50 mL). To this was addedPd(dppf)₂Cl₂ (10 mg, 0.012 mmol) followed by 2M Na₂CO₃ (8 mL). Themixture was then heated at reflux for 6 hrs. After a cooling to roomtemperature the solids were filtered off and the residue evaporated. Thecrude product was purified by flash chromatography (EtOAc:MeOH) to givePI3KI1. LRMS-LC/MS (m/z): [M+H]⁺ calcd for C₂₇H₂₀F₂N₄O₄S, 534.1; found535.1).

SUH78017 (50 mg, 0.094 mmol) and compound 9 (32.7 mg, 0.094 mmol) wasdissolved in DMF (1 mL) and stirred. Progress of the reaction wasmonitored by analytical LRMS-LCMS. Following the completion of thereaction crude product was purified by preparative RP-HPLC [A=2 mMammonium acetate buffer (pH 7.0), B=acetonitrile, solvent gradient 5% Bto 80% B in 35 min] to yield 80% of compound 10. LRMS-LC/MS (m/z):[M+H]⁺ calcd for C₃₅H₂₇F₂N₅O₆S₃, 747.1; found 748.1)

FAP-PI3KI1

Compound 10 (22.3 mg, 0.019) and compound 11 (10 mg, 0.018 mmol) wasdissolved in anhydrous DMSO and stirred under inert atmosphere. Progressof the reaction was monitored by analytical LRMS-LCMS. Following thecompletion of the reaction crude product was purified by preparativeRP-HPLC [A=2 mM ammonium acetate buffer (pH 7.0), B=acetonitrile,solvent gradient 5% B to 80% B in 35 min] to yield 80% of the finalproduct FAP-PI3KI1. LRMS-LC/MS (m/z): [M+H]⁺ calcd for C₅₂H₄₈F₄N₁₀O₁₂S₃,1177.2 found 1179.1).

Cell Culture and Animal Husbandry

IPF patient cell lines were kindly donated by Dr. Ivan O. Rosas, M.D.Brigham and Women's Hospital, Boston, Mass. C57BL6/6-NCrl (Strain code:027) mice were purchased from Charles River and maintained on normalrodent chow. They were housed in a sterile environment on a standard 12h light-and-dark cycle for the duration of the study. All animalprocedures were approved by the Purdue Animal Care and Use Committee(PACUC) in accordance with NIH guidelines.

In Vitro Studies

Live Cell Imaging of FAP-FITC Internalization

HLF1-hFAP cells were seeded in the glass-bottom dish and incubated withadequate amount of endosomes tracker (Rab7a-RFP, ThermoFisher) forovernight. Then cells were incubated with FAP-FITC (10 nM) for 1 hour at4° C. Followed by staining with 5 nM DRAQ5 nucleus dye (ThermoFisher)and 3 times of PBS washes, spatial localization of FAP-FITC was monitorat given time under ambient temperature by confocal microscope (FV 1000,Olympus). Confocal images were further processed by FV10-ASW, Olympussoftware.

Immunofluorescence of FAP and αSMA Expression in Fibroblasts

HLF1 cells, primary IPF fibroblasts and the non-IPF fibroblasts werecultured, fixed, and permeabilized on glass-bottom dishes forimmunofluorescent staining. Primary antibodies against hFAP (1:200,FAB3715R, R&D Systems) or αSMA (1:1000, ab21027, Abcam) were incubatedovernight at 4° C. After PBS washes, incubated with secondary antibodyof Alexa Fluor® 488 anti-Goat antibodies (Abcam, 1:400). Images werecaptured and analyzed by confocal microscope.

Sirius Red Staining for Secreted Total Collagen

Confluent HLF1 cells were seeded in DMEM medium containing 10% FBS andthen 0.4% serum starvation overnight before stimulation of collagensecretion. TGFb1 (0.1 ng/ml) were added into cells with or without PI3Kinhibitors. At 2 days post co-incubation, culture medium was collectedfor determination of total secreted collagen level. Total collagen levelwas determined by Sirius Red Total Collagen Detection Kit (Chondrex,Inc). Basically, concentrated sample incubated with 500 ml of Sirius RedSolution with for 20 minutes at room temperature. Pellet was collect bycentrifuge at 10,000 rpm for 3 minutes and followed by washing with 500ml of washing solution for 3 times. Add 250 ml of Extraction Buffer tothe Sirius Red stained pellet and read the OD at 510-550 nm.

Western Blot Analysis of Cultured Fibroblasts

Serum starved confluent HLF1 cells were co-incubated in mediumcontaining 1 ng/ml TGFb1 with or without designate concentrations ofPI3K inhibitors for 24 hours. Cells were harvested and lysed for Westernblot analysis. Following sodium dodecyl sulphate polyacrylamide gelelectrophoresis and blocking, membranes were incubated with antibodiesto detect pSMAD2^(Ser465/467) (#3101, Cell Signalling Technology), orpAkt^(Ser473) (#4060, Cell signalling Technology), and signalsvisualized with ECL Western Blotting Detection Reagents (GE Healthcare).Following stripping, membranes were blocked and re-probed withantibodies specific for total SMAD2 (#3103, Cell Signalling Technology)or total Akt (#4060, Cell Signalling Technology).

In Vivo Studies

Bleomycin-Induced Lung Fibrosis Model

8 to 10-week old C57BL/6-NCrl (Strain Code: 027) male mice (CharlesRiver) were anesthetized (mixture of xylazine/ketamine), followed by asingle intratracheal injection of freshly prepared 0.75 mg/Kg ofbleomycin sulfate (Cayman Chemicals, Cat N13877) in 50 μL of sterilephosphate-buffered saline (PBS). Control mice were injected with 50 μLof sterile PBS. Body weights were monitored throughout the study. Toaccess the FAP expression and fibrosis in the longitudinal study thelungs were harvested at Days 7, 14 and 21 post-bleomycin instillation.For the therapy with FAP-PI3K inhibitor, same procedure was carried outto administer bleomycin and the lungs were harvested at Day 21 to accessthe therapeutic efficacy (Day 0 was accounted as the day of bleomycinadministration).

Hydroxyproline Assay

Total lung collagen was determined by analysis of hydroxyproline aspreviously described.³⁰ The right lung was consistently dedicated forthis assay to allow comparison. Briefly, harvested right lung washomogenized in PBS (PH 7.4), digested with 12N HCl at 120° C. for 3 hr.Citrate/acetate buffer (PH 6.0) and Chloramine-T solution were added atroom temperature for 20 minutes and the samples were incubated withEhrlich's solution for 15 min at 65° C. Samples were cooled to roomtemperature and read at 550 nm. Hydroxyproline standards (Sigma, MO) atconcentrations between 0 to 100 μg/ml were used to construct a standardcurve.

Histopathological Evaluation of Pulmonary Fibrosis

The left lung was inflated and fixed with 4% paraformaldehyde. Lungtissues were embedded in paraffin, and 10-μm sections were prepared andstained using H&E and Masson's Trichrome. The severity ofbleomycin-induced fibrosis was determined by semiquantitativehistopathological scoring, at the indicated dates after bleomycinadministration.²⁹

In Vivo Fluorescence Imaging

Mice were treated via tail vein (i.v.) injection with 5 nmol of FAPtargeted NIR dye conjugate (FAP-S0456) and imaged 2 hr post-injectionusing a Spectral AMI optical imaging system. For competitionexperiments, a 100-fold excess of base the FAP ligand was used. Thesettings were as follows: Object height, 1.5; excitation, 745 nm;emission, 790 nm; FOV, 25; binning, 2; f-stop, 2; acquisition time, 1 s.After the completion of whole-body imaging, animals were dissected, andselected organs were collected and imaged again for completebiodistribution. The conditions remained same for the longitudinalimaging study, except the mice were imaged at day 7, day 14, and day 21post-bleomycin administration.

In Vivo Micro-CT Imaging

Micro-CT analysis of the whole lung was performed at day 7, day 14, andday 21 post-bleomycin administration Briefly animals were anesthetizedwith isoflurane and fixed in prone position. Micro-CT images wereacquired on a Quantum FX micro-CT system (Perkin Elmer, Waltham, Mass.)with cardiac gating (without respiratory gating), using the followingparameters: 90 kV; 160 μA; FOV, 60×60×60 mm; spatial resolution, 0.11mm, resulting in a total acquisition time of 4-5 minutes

Example 1. Design and Synthesis of FAPL-FITC Conjugate for Analysis ofFAP Targeting

In this Example, an imaging agent conjugate comprising FAP targetingligand (FAPL) and a fluorescein such as FITC was generated according tothe scheme shown in FIG. 1.

In order to determine the conjugate's in vitro binding features such asbinding affinity to FAP, and its bio distribution within FAP expressingcells, FAPL-FITC conjugate was incubated with a FAP transfected cellline HLF1 (human fibroblast cells), confocal microscopy and flowcytometry are used to observe the conjugate's specific targeting to FAPexpressing cell line, and its subsequent endocytosis in the cell line.See FIG. 2 and its legend, which shows FAPL-FITC binds well with goodcompetition in the hFAP-HLF1 cell line. In addition, FAP ligand canrecognize and target FAP with good specificity. FAPL_FITC conjugates areinternalized upon receptor engagement. This shows the conjugate islikely to deliver a payload of effectors to the activated fibroblast,for example, the imaging agent or, a therapeutic drug.

Example 2. Binding of FAPL-FITC by Human IPF Patient Cell Line

In this Example, human IPF patient cell line and non-IPF control cellline were stained with FAP antibody and αSMA antibody respectively.Confocal microscopy indicates both FAP and αSMA are predominantlyexpressed in IPF lung fibroblasts. See FIG. 3A. When the IPF patientcell line is incubated with FAPL_FITC, the flow cytometry analysis showsFAPL_FITC stained samples in FIG. 3B.

Example 3. Design, Synthesis of PI3KI1 and FAP-PI3KI1

In this Example, we provided a novel pan PI-3Kinase-mTOR inhibitor namedPI3KI1. This potential IPF drug has a good handle to incorporatereleasable linkers to conjugate to a targeting ligand, for example, aFAP ligand in example 2-3.

The design and synthesis of the novel pan PI-3Kinase-mTOR inhibitor isshown in FIG. 4.

The synthesis scheme of novel FAP targeted pan-Pi3K inhibitor(FAPL_PI3KI1) is shown in FIG. 5.

Example 4. Evaluation of Myofibroblast Inactivation with FAPL-TargetedPI-3 Kinase Inhibitor In Vitro

In this Example, we have shown that the novel PI-3Kinase inhibitorPI3KI1 inhibits Akt phosphorylation better than its GSK counterpartOMIPALISIB. The latter is in a clinical trial for IPF therapeutic drug.The PI3KI1 also suppresses collagen secretion and collagen gelcontraction in HLF-1 cell line. Because the novel PI-3Kinase inhibitorPI3KI1 has a free hydroxyl group, it allows facile conjugation to a FAPligand, show in the example 2 and 3. The novel drug does not havetoxicity and it behaves similarly to GSK drug OMIPALISIB. See FIG. 6 andits Legend.

Example 5. Ex Vivo IPF Patient Cell Line Data

In this Example we established that FAP targeted PI-3 kinase inhibitorFAP_PI3KI1 suppresses TGFβ induced collagen secretion at low drugconcentration, better than free PI3KI1 and free GSK drug OMIPALISIB inIPF patient cells. See FIG. 7 and its legend.

Example 6. Evaluation of FAP Targeting in Mouse Fibroblast Cell Line InVitro

In this Example, we have shown that FAPL_FITC can recognize mouse FAP.Briefly, NIH-3T3 cells were induced with TGFβ to express FAP (mimickingthe pathogenesis of fibrosis in mice). FAPL_FITC can recognize almost98% of the cell population that binds to a specific monoclonal antibodyagainst mouse FAP. See FIG. 8 and its legends.

Example 7. Evaluation of FAP Targeting of IPF Lung in Mouse Model of IPFIn Vivo

In this Example, FAPL conjugated with drug or other effectorspecifically target fibrotic lungs in mice day 14 after acute lunginjury. Briefly, both the saline treated and fibrotic lungs with FAPcompetition showed minimum retention of the NIR signal while thediseased lung without competition showed high uptake of the FAP_S0456dye. Both micro CT and NIR imaging at different time points (day 7, 14,21) after acute lung injury, showed progression of fibrosis.

In correlation with the hydroxyproline and immunostaining data, it isshown that highest optical intensity was achieved at day 14, indicatingpeak fibrosis, and slowly subside around day 21. See FIG. 9 and itslegends.

Example 8. Evaluation of Myofibroblast Inactivation with FAPL-TargetedPI-3 Kinase Inhibitor In Vivo

In this Example, attenuation of fibrosis was demonstrated with theincreased survival rate, and the reduction of hydroxyproline in theFAPL_PI3KI-SUH treated mice over the untreated mice. See FIG. 10 and itslegends.

Example 9. Imaging Distribution of Lung Fibrosis

In this example, Radio-imaging of experimental lung fibrosis is shown inFIG. 19 A (in various organs) and in FAP targeted ^(99m)TC in the lungs(FIG. 19B).

1. A conjugate to target Fibroblast Activation Protein (FAP) expressingcells in fibrotic lung diseases, comprising a targeting ligand to FAP(TL), a linker (L) and an effector (E), wherein said TL has a molecularweight below 10,000, said L is a non-releasable linker when saideffector is an imaging agent or a radioactive therapeutic agent, or areleasable linker when said effector is a therapeutic drug, wherein saidlinker is selected from the group consisting of a pegylated, alkyl,sugar or a peptide based dual linker.
 2. The conjugate according toclaim 1, wherein said imaging agent is a fluorescent molecule.
 3. Theconjugate according to claim 2, wherein the fluorescent molecule isfluorescein and the conjugate (FAP-FITC) has the structure of


4. The conjugate according to claim 2, wherein the fluorescent moleculeis S0456 and the conjugate has the structure of


5. The conjugate according to claim 2, wherein the fluorescent moleculecomprising the structure of


6. The conjugate according to claim 1, wherein the effector is a PETimaging agent.
 7. The conjugate according to claim 6, wherein the PETimaging agent contains the structure of


8. The conjugate according to claim 1, wherein the effector is animaging or therapeutic agents containing DOTA, NOTA, TETA or NODAGAchelating agents


9. The conjugate according to claim 1, wherein the effector is a 99mTcimaging agent comprises the structure of


10. The conjugate according to claim 1, wherein the radioactivetherapeutic agent contains the structure of


11. The conjugate according to claim 1 wherein the TL is a smallmolecule comprising the structure of

wherein X is

wherein R₁ and R₂ are the same or different, and are each independentlyselected from the group consisting of hydrogen, halogen and C₁-C₄ alkyl;R₃ is a C₁-C₄ alkyl, nitrile, or isonitrile; R₄ is H or —CH₃; R₅ and R₆are the same or different, and are each independently selected from thegroup consisting of hydrogen, halogen, and C₁-C₄ alkyl; and R₇-R₉ arethe same or different, and are each independently selected from thegroup consisting of hydrogen, methoxy, halogen, CF₃ and C₁-C₄ alkyl. 12.The conjugate of claim 11, wherein each of R₁ and R₂ is a halogen. 13.The conjugate of claim 11, wherein each of R₁ and R₂ is fluorine. 14.The conjugate according to claim 1, wherein the effector is a kinaseinhibitor for VEGFR1, VEGFR2, VEDFR3, FGFR1, FGFR2, or PDGFR.
 15. Theconjugate according to claim 1, wherein the effector is a kinaseinhibitor for FAK or ROCK.
 16. The conjugate according to claim 1,wherein the effector is an SMAD inhibitor.
 17. The conjugate accordingto claim 1, wherein the effector is a cytotoxic agent.
 18. The conjugateaccording to claim 1, wherein the effector is a pan PI-3 kinase/mTORinhibitor.
 19. A panPI-3 Kinase/mTOR inhibitor comprising the structureof (PI3KI1):


20. The conjugate according to claim 1, having the structure of


21. The conjugate according to claim 18, wherein the PI-3 kinaseinhibitor comprising the structure below:

wherein X can be any of the following


22. The conjugate according to claim 1, wherein the targeting ligand toFAP has a binding affinity to FAP in the range between about 1 nM andabout 20 nM.
 23. A method of diagnosing IPF in a subject, comprising thefollowing steps: obtaining the lung tissue from the subject, whereinsaid tissue may or may not express FAP in fibroblast cells; providing tothe tissue with a conjugate of TL-L-I, wherein TL is a targeting ligandA conjugate to target Fibroblast Activation Protein (FAP) expressingcells in fibrotic lung diseases, wherein said TL has a molecular weightbelow 10,000, said L is a non-releasable linker, said I is an imagingagent; and identifying imaging illustrated fibroblast cells as FAPexpressing activated fibroblast cells as the hallmark of IPF. 24.(canceled)
 25. A method of treating IPF in a subject, comprising thesteps of: providing to the IPF patient cells with a pharmaceuticallyeffective amount of conjugate of TL-L-D, wherein TL is a targetingligand to FAP that has a molecular weight below 10,000, L is areleasable linker and D is a therapeutic drug that has pan PI-3Kinaseinhibitory effect; and monitoring lung tissue extracellular matrixdeposit amount reduction upon the treatment of TL-L-D. 26.-33.(canceled)